Search

Much progress has been made in the management of chronic-phase chronic myeloid leukemia (CP-CML), but there is a continuing imperative to develop curative treatments, predict patient responses to specific modalities, and anticipate disease relapse or progression. These needs underlie continuing interest in methods to detect and quantify the relevant leukemic cells in clinical samples with improved reliability and specificity. We report the results of comparing three methods to enumerate primitive CP-CML cells in the same samples: genotyping CD34+38− cells directly by fluorescence in situ hybridization, and measuring BCR-ABL1 transcript-genotyped colony-forming cell outputs in either 5-week long-term cultures (LTCs) containing non-engineered mouse fibroblasts or in 6-week LTCs containing mouse fibroblasts engineered to produce human Steel factor, granulocyte colony-stimulating factor, and IL-3. The results demonstrate that the first two methods significantly overestimate the prevalence of primitive CP-CML cells by comparison to the third. In additional studies, we found that CML-CD34+ cells can repopulate the marrow and spleen of serially transplanted adult NOD/SCID-IL-2Rγ chain-null mice for more than 1 year with an almost exclusive myeloid differentiation in primary and secondary recipients and without evidence of disease progression. These findings underscore the importance of long-term functional in vitro and in vivo endpoints to identify and characterize CP-CML stem cells. [Copyright &y& Elsevier]

Summary: The JAK2V617F mutation is associated with distinct myeloproliferative neoplasms, including polycythemia vera (PV) and essential thrombocythemia (ET), but it remains unclear how it generates disparate disorders. By comparing clonally-derived mutant and wild-type cells from individual patients, we demonstrate that the transcriptional consequences of JAK2V617F are subtle, and that JAK2V617F-heterozygous erythroid cells from ET and PV patients exhibit differential interferon signaling and STAT1 phosphorylation. Increased STAT1 activity in normal CD34-positive progenitors produces an ET-like phenotype, whereas downregulation of STAT1 activity in JAK2V617F-heterozygous ET progenitors produces a PV-like phenotype. Our results illustrate the power of clonal analysis, indicate that the consequences of JAK2V617F reflect a balance between STAT5 and STAT1 activation and are relevant for other neoplasms associated with signaling pathway mutations. [ABSTRACT FROM AUTHOR]

The critical human cells that produce neutrophils and platelets within 3 weeks in recipients of hematopoietic transplants are thought to produce these mature blood cells with the same kinetics in sublethally irradiated immunodeficient mice. Quantification of their numbers indicates their relative underrepresentation in cord blood (CB), likely explaining the clinical inadequacy of single CB units in rescuing hematopoiesis in myelosuppressed adult patients. We here describe that exposure of CD34 + CB cells ex vivo to growth factors that markedly expand their numbers and colony-forming cell content also rapidly (within 24 hours) produce a significant and sustained net loss of their original short-term repopulating activity. This loss of short-term in vivo repopulating activity affects early platelet production faster than early neutrophil output, consistent with their origin from distinct input populations. Moreover, this growth factor-mediated loss is not abrogated by published strategies to increase progenitor homing despite evidence that the effect on rapid neutrophil production is paralleled in time and amount by a loss of the homing of their committed clonogenic precursors to the bone marrow. These results highlight the inability of in vitro or phenotype assessments to reliably predict clinical engraftment kinetics of cultured CB cells. [ABSTRACT FROM AUTHOR]

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo . However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34 + cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1 –/– -IL2Rγc –/– (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD- Prkdc scid/scid - IL2Rγc –/– (NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W 41 /W 41 derivatives showed superior chimerism in the W 41 -deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemias (AML) comprise a genetically diverse group of human hematologic malignancies with a generally poor prognosis and modes of pathogenesis that have been difficult to investigate. Historic evidence and more recent findings have identified hematopoietic stem cell programs as major targets of mutations that predispose to and/or initiate a multistep process of transformation. However, details of the changes involved and how they interact in emerging human leukemic cells have been particularly challenging to characterize. This is due to an emerging appreciation of the heterogeneity in events that impact the differentiation processes of normal human hematopoietic cells, as well as difficulties in recreating the full leukemogenic alterations in the actual human cells affected, to avoid discrepancies inherent in studying model organisms. Here we describe several models of de novo human leukemogenesis that illustrate their power and the novel results they can generate. These include our recent discovery of a novel "latent" leukemic state that can be obtained in MYC-transduced human cord blood cells regenerating a normal spectrum of lymphoid and myeloid progeny in transplanted immunodeficient mice, but that can then be rapidly activated to an aggressive form of AML by in vivo exposure to a single human growth factor. These findings portend the future utility of de novo human leukemogenesis models as new platforms for elucidating shared molecular mechanisms responsible for different stages of human leukemogenesis that may be initiated by different mutations or exposure to different microenvironmental conditions. The flexibility and consistency of these models also make them attractive for identifying and testing new treatment strategies targeting mechanisms required for disease manifestation. [ABSTRACT FROM AUTHOR]

Inactivating mutations in IKZF1 , the gene that encodes the transcription factor IKAROS, are recurrent in poor-prognosis human B-cell leukemias, in which these mutations co-exist with BCR-ABL1 or other genetic changes that activate similar intracellular signaling pathways. However, little is known about the mechanism(s) by which loss of IKAROS activity may co-operate with BCR-ABL1 to transform lymphoid cells. To investigate this question, we used expression of a dominant-negative isoform of IKAROS (IK6) to suppress endogenous IKAROS activity in the interleukin-3 (IL-3)-dependent mouse pro-B BA/F3 cell line and in an IL-3-independent BCR-ABL1 + derivative. We then used intracellular phospho-flow cytometry to assess the effects of BCR-ABL1 and IK6, alone and in combination, on the signaling state of the cells before and after their stimulation with IL-3. BCR-ABL1 and IK6 each produced a constitutively activated signaling phenotype and also enhanced the signaling responses of BA/F3 cells to IL-3. These effects, however, were neither equivalent nor additive, and IK6 alone was insufficient to confer the IL-3-independent growth characteristic of BCR-ABL1 + BA/F3 cells. In addition to its effects on lymphoid cells, IK6 also induced constitutively activated signaling in a subset of myeloid leukemia cell lines. Together, these studies indicate an ability of IK6 to enhance intracellular signaling in both lymphoid and myeloid cells, but not to synergize with BCR-ABL1 in this model system. [ABSTRACT FROM AUTHOR]